1. Field of the Invention
The present invention relates to the fields of pharmacology and treatment of diseases, particularly gastrointestinal dysfunction. More specifically, the present invention discloses in one aspect the acceleration of gastric emptying by tetrahydrobiopterin (BH4) and derivatives thereof.
2. Description of the Related Art
Gastroparesis is a devastating disease affecting predominantly young women, with a female:male ratio of 4:1 (Soykan et al., 1998). Although a variety of diseases are associated with gastroparesis, the two most common subtypes are diabetes and idiopathic diabetic gastropathy (Camilleri., 2002; Bityutskiy et al., 1997), a syndrome of delayed gastric emptying leading to nausea, vomiting, postprandiol fullness, abdominal pain and early satiety. Because of its chronic and often intractable nature, the disorder has a tremendous impact on both patients and society (Bell et al., 2002; Revicki et al., 2004). Long standing and poorly-controlled diabetes results in the disturbance of several gastric functions such as gastric myoelectric activity, antroduodenal motor activity, gastric emptying and gastric visceral sensation (Parkman et al., 2004). Although delayed gastric emptying has long been taken as a hallmark of this condition, in recent reports most experts concur that this correlates poorly if at all with clinical symptoms (Rayner and Horowitz, 2005; Parkman et al., 2004). The biggest barrier to the development of effective therapy for gastroparesis has been the lack of understanding of its pathogenesis and/or pathophysiology. Consequently, treatment has been empirical and only partially effective, if at all, in relieving major symptoms.
Normally, gastric motility is regulated in large part by neurons of the enteric nervous system located in the muscle wall (Camilleri, 2002). These neurons are either excitatory (releasing acetylcholine) or inhibitory (releasing nitric oxide and vasoactive intestinal peptide). Nitric oxide (NO) is the principal non-adrenergic non-cholinergic (NANC) inhibitory neurotransmitter in the gastrointestinal tract and is produced by neuronal NOS, expressed in inhibitory enteric neurons (Coleski et al., 2005; Ishiguchi et al., 2000; Mizuta et al., 1999; Martinez-Cuesta, 1995; Takahashi, 2003; Nakamura, 1998; Nakao et al., 1998). NO activates soluble guanylate cyclase (sGC), producing an increase in the intracellular cyclic guanosine-3′, 5′-monophosphate (cGMP), leading to muscle relaxation (Takahashi, 2003; Patil et al., 2005; Kim et al., 2003; Shah et al., 2000). Nitrergic signaling is particularly responsible for gastric accommodation and pyloric relaxation in response to a meal. The importance of NO in gastric function was established by the findings of pyloric hypertrophy and gastric dilation in mice with a targeted genomic deletion of neuronal nitric oxide synthase (nNOS−/−) (Huang et al., 1993; Mashimo et al., 2000). Vagal modulation of enteric neuronal function (both inhibitory and excitatory) also plays an important role in gastric physiology and is predominantly cholinergic in character (Undeland et al., 1997; Undeland et al., 1996; Li and Owyang, 2003).
Expression of nNOS is distinguished by a remarkable diversity. Different 5′ mRNA variants of nNOS are reported in various tissues including the gut (Huber et al., 1998; Saur et al., 2000; Putzke et al., 2000; Saur et al., 2002; Panda et al., 2003). 5′ mRNA variants of nNOS are generated either by alternative promoter usage resulting in different mRNA that encode for the same protein (nNOS alpha, 155 KDa) or alternative splicing encoding NH(2)-terminally truncated proteins (nNOS beta and gamma) that lack the PDZ/GLGF domain for protein-protein interaction (Saur et al., 2002; Panda et al., 2003; Saur et al., 2000; Kone et al., 2003). nNOS mutant mice, in which exon 2 (encoding for the PDZ/GLGF motif) and, consequently, full length nNOSalpha, was disrupted, maintain some nNOS expression due to presence of nNOSbeta and nNOSgamma. However, gastric function is severely affected with delayed gastric emptying (Huang et al., 1993; Mashimo et al., 2000). These studies suggest that nNOSalpha, but not other proteins, are essential for normal gastric motor function. The molecular mechanisms responsible for impaired NO function in diabetes remains incompletely understood with both a decrease (Watkins et al., 2000; Wrzos et al., 1997) and an increase (Adeghate et al., 2003) in nNOS expression being reported in the literature.
In diabetic gastric dysfunction, antral motility and the co-ordination of pressures between antrum and duodenum are diminished. Antral hypomotility has been recorded with intraluminal transducers in patients with diabetes mellitus. Abnormal gastrointestinal motility in diabetes mellitus is likely multifactorial in origin, reflecting disturbances in enteric and vagal neural activity as well as interstitial cells of Cajal (ICC) and smooth muscle function. Of these, enteric neuropathy may be particularly important (Martinez-Cuesta et al., 1995; Belai et al., 1985; Belai and Burnstock, 1987; Burnstock et al., 1988; Belai and Burnstock, 1990; Belai et al., 1991; Belai et al., 1988). Several studies of animal models of diabetes have convincingly shown disturbances in enteric nerves, particularly involving nitrergic nerves (Cellek., 2004; Cellek et al., 2005; Cellek et al., 2003; Buchan, 1990; Di Giulio et al., 1989). Impairment in nitrergic relaxation resulting from either neuronal loss or dysfunction may contribute to gastropathy in both streptozotocin (STZ) induced diabetes (Watkins et al., 2000; Jenkinson and Reid, 1995) as well as spontaneously diabetic male rats and mice (Takahashi et al., 1997). These disturbances provide a rational pathophysiological explanation for observations of decreased gastric compliance and pyloric relaxation noted in diabetic patients. In the absence of effective nitrergic output to muscle, gastric accommodation is impaired, resulting in early satiety and discomfort. Further, a functional obstruction at the gastric outlet due to a non-relaxing pylorus leads to delayed emptying (James et al., 2004; Cellek et al., 2003; Tougas et al., 1992; Ishiguchi et al., 2002). Diabetic rats and mice show defects in nitrergic relaxation and nNOS expression before neuronal degeneration in the pyloric sphincter and this was reversed by insulin treatment (Watkins et al., 2000; Cellek et al., 2003).
Several co-factors are known to be important for nNOS activity, including NADPH, calcium and tetrahydrobiopterin (BH4). Tetrahydrobiopterin regulates the homodimeric conformation of all three isoforms of NOS [endothelial(e)NOS; inducible(i)NOS; neuronal(n)NOS] (Cosentino and Luscher, 1999). BH4 also serves as a cofactor for three aromatic amino acid hydroxylases:phenylalanine (PAH), tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH). Additionally, BH4 is scavenger of oxygen-derived free radicals. BH4 has been clinically investigated as therapy for phenylketonuria (PKC), Parkinson's disease, dystonia, depression, Rett Syndrome, infantile autism, senile dementia, Alzheimer's disease and atherosclerosis. Lack of BH4 biosynthetic genes causes several abnormalities in mice. Incubation with saturating concentrations of tetrahydrobiopterin induces substantial conformational changes in the homodimeric structure of nNOS, yielding a stabilized nNOS dimer with maximal NO-producing activity (Gorren et al., 1996; Klatt et al., 1995). In mice, the highest levels of tetrahydrobiopterin are found in the liver, adrenals and stomach (Kobayahi et al., 1991). Tetrahydrobiopterin synthesis occurs via two distinct pathways: a de novo synthetic pathway which uses GTP as a precursor and a salvage pathway for preexisting dihydropterins (Thony et al., 2000; Gross and Levi, 1992) (FIG. 1).
GTP cyclohydrolase 1 (GTPCH1) is the rate-limiting enzyme for tetrahydrobiopterin de novo pathway leading to synthesis of dihydroneopterin triphosphate. Treatment of HEK293 cells with 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTPCH1 leads to depletion of tetrahydrobiopterin, destabilization of the dimeric form of nNOS and enhanced ubiquitinylation of nNOS (Kamada et al., 2005). However, addition of sepiapterin, a precursor of tetrahydrobiopterin in the salvage pathway, completely reverses the effect of DAHP on nNOS destabilization (Kamada et al., 2005; Shang et al., 2005). In the absence of tetrahydrobiopterin, uncoupling of NO production occurs and electron flow from the reductase domain to the oxygen domain of nNOS is diverted to molecular oxygen rather than L-arginine. This leads to super oxide production; super oxide in turn not only degrades NO, but also forms peroxynitrite a potent oxidant that can rapidly oxidize BH4 to BH3+ and subsequently to BH2. BH2 may compete with tetrahydrobiopterin for nNOS binding, resulting in further impaired nNOS bioactivity.
There is considerable evidence supporting an important role for impairment in the tetrahydrobiopterin biosynthetic pathway in mediating dysfunction of NOS isoforms such as eNOS both in vivo and in vitro. DAHP, a GTPCH1 inhibitor reduces the sensitivity to acetylcholine (endothelium-dependent)-induced vascular relaxation (mediated by NO) in normal mice and this inhibitory effect was shown to be restored by addition of tetrahydrobiopterin in vitro (Pannirselvam et al., 2002). Treatment of diabetic vascular endothelial cells with sepiapterin (the tetrahydrobiopterin precursor in the salvage pathway, (FIG. 1), significantly improves NO synthesis. Preincubation of vascular rings with either tetrahydrobiopterin or sepiapterin enhances Ach(acetylcholine-)-induced relaxation in diabetic mice (Cosentino and Katusic, 1995; Mitchell et al., 2003; Pieper, 1997; Tsutsui et al., 1996). In addition, dietary supplementation of sepiapterin increases ACh-induced vascular relaxation in diabetic mice (Pannirselvam et al., 2003). In cultured endothelial cells exposed to high glucose (Cai et al., 2005), ex vivo gene transfer of GTPCH1 restores eNOS dimerization, attenuates impaired endothelium-dependent relaxation and increases NO production (Cai et al., 2005; Meininger et al., 2004). Selectively augmenting endogenous tetrahydrobiopterin levels by targeting over expression of GTPCH in endothelial cells in vivo preserves eNOS dimerization in streptozotocin (STZ)-induced diabetes mice (Cai et al., 2005; Alp et al., 2003). The beneficial effects of tetrahydrobiopterin supplementation in reversing impaired endothelium dependent relaxation have also been demonstrated in human patients. BH4 therapy was shown to be useful in improving endothelium-dependent relaxation in patients with hypercholesteromia (Stroes et al., 1997), venous conduits used for coronary artery bypass graft surgery (Maier et al., 2000), patients with type II diabetes (Heitzer et al., 2000), normal epicardial arteries (Setoguchi et al., 2001) and smokers (Heitzer et al., 2000).
Furthermore, there is increasing evidence of gender-related differences in gastric emptying. The effect of gender in a healthy population on gastric emptying remains controversial though it appears that women may have slower solid and liquid emptying. Ambulatory antroduodenal manometry has shown shorter migrating motor complex (MMC) periods in women compared to men (Aytug et al., 2001). The mechanisms responsible for these differences are not completely understood. In a recent study of duodenojejunal motility, women in the follicular phase were found to exhibit motor activity similar to that of men (Soffer et al., 1998). On the other hand, another study demonstrated attenuated postprandial antral contractile activity in the follicular phase of women compared to men (Knight et al., 1997). Additionally, animal studies demonstrated that the gastric emptying rate was slower in ovary-intact female rats compared to ovariectomized (depletion of ovarian hormones; estrogen and progesterone) female rats (Chen et al., 1995; Coskun et al., 1995). Furthermore, studies suggested that estradiol-17β (E2) but not progesterone (P4) may be responsible for delayed gastric emptying and increased nitrergic system. In addition to this, studies suggest that P4 treatment decreased the resting tension fundus, inhibited mean contractile amplitude of antrum and the motility index of pylorus in rats (Wang et al., 2003). Diabetes induction decreases both the circulatory E2 and P4 levels in both women and female rats (Resnick and Howard, 2002; Mankhey et al., 2005; Leonelli et al., 2007; Veiga et al., 2006; Caruso et al., 2007).
Sex steroid hormones mediate their biological actions through their respective nuclear (genomic) cytoplasmic/membrane (non-genomic, rapid via nitric oxide elevation) receptors (Boonyaratanakornkit and Edwards, 2007). Estrogen receptors (ERs) and progesterone receptors (PRs) are expressed as two proteins: ERα and ERβ, and PR-A and PR-B. ERα and ERβ are expressed from two different genes, whereas PR isoforms are produced from alternate use of two promoters from the same gene. Sex steroid hormone receptors require both a ligand (sex hormones, insulin, growth factors etc) and interactions with other proteins, such as coregulators, to achieve maximal transcriptional activation of genes. Female sex steroids (both E2 and P4) has multiple beneficial actions that includes neuroprotection, maintaining glucose homeostasis in both health and diabetes (Leonelli et al., 2007; Caruso et al., 2007; Boonyaratanakornkit and Edwards, 2007; Barros et al., 2006). In particular, estrogen has both genomic and rapid nongenomic effects via its receptors on vascular endothelium, including activation of NO synthesis (Widder et al., 2003; Cid et al., 2002). Previous studies demonstrated that nNOS is involved in estrogen mediated neuroprotection in neuroblastoma cells (Wen et al., 2004; Murphy and Steenbergen, 2007; Scordalakes et al., 2002; Garcia-Duran et al, 1999; Gingerich and Krukott, 2005; E1-Sakka et al., 1999). The role of progesterone and its metabolites via PR's on NO mediated cardioprotection has been recently reported in postmenopausal women (Simoncini et al., 2007). Upon binding to their respective receptors, sex steroids, utilizes several cell signaling mechanisms such as cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K)/Akt pathways, for their actions. In the genomic pathway, sex steroids binds to their cytosolic/nuclear receptors, leading to activation of MAPK/Akt, increase gene transcription and upregulate nitric oxide production. In non-genomic pathway, sex steroids binds to their membrane receptors, which are coupled to increased Ca2+ release from the endoplasmic reticulum, and stimulate MAPK/Akt/PI3K pathway, leading to NO production. NO diffuses into the smooth muscle cells, binds to adenylate cyclase (AC) or guanylate cyclase (GC) and increases cAMP or cGMP respectively. Significant actions for sex steroids have been noticed in the gastrointestinal tract in various experimental animal models and human clinical settings (Kawano et al., 2004). Shah et al studies demonstrated that estrogen treatment increases nNOS positive neurons in the female rat stomach. Several studies demonstrated that both ERs are primarily localized in nerve cells of the gut (Shah et al., 2001; Kawano et al., 2004; Campbell-Thompson et al., 2001).
Estrogen treatment elevates both the expression of GTPCH1 and BH4 levels in rat brain neurons through estrogen receptors (Serova et al., 2006; Serova et al., 2004; Lam et al., 2006). In vitro hyperglycemia decreases both BH4 biosynthesis and nitric oxide and estrogen supplementation restored this effect via ERα in bovine aortic endothelial cell culture (Miyazaki-Akita et al., 2007). Diabetes induction decreases the circulatory estrogen and progesterone levels in both women and female rats. Previous reports demonstrated that estrogen receptors are localized in stomach enteric neurons. The beneficial role for E2 treatment on both GTP cyclohydrolase1 (GTPCH1) expression and nNOS expression has been well demonstrated.
Despite this, the prior art is deficient in the role played by tetrahydrobiopterin or derivatives thereof in the nitric oxide induced diabetic gastroparesis. Additionally, the prior art is also deficient in understanding the gender-related differences in gastric emptying. The present invention fulfills this long-standing need and desire in the art.